SUMMARY The R03 project described here focuses on a naturally occurring p53 isoform, called ?Np53, with well- established links to mammalian aging. The ?Np53 isoform was first characterized with mouse knock-in studies. Expression of ?Np53 together with WTp53 caused accelerated aging, accompanied by age-related pathologies such as osteoporosis and memory loss. Importantly, expression with WTp53 is required for the accelerated aging phenotype, indicating that mixed ?Np53:WTp53 tetramers are essential. This presents a significant barrier to understanding how ?Np53 specifically affects cellular function, because co-expression of ?Np53 and WTp53 will result in tetramer heterogeneity, including formation of native, WTp53 tetramers. To circumvent this problem, we expressed ?Np53:WTp53 as a single transcript that ensured a 2:2 stoichiometry (?Np53:WTp53) within the tetramer. This circumvented confounding issues associated with DNp53 and WTp53 co-expression and allowed clear delineation between cellular effects triggered by WTp53 vs. DNp53:WTp53. In a proof of concept study (published 2013), we confirmed that this tethering strategy would yield reliable, biologically relevant data. The experiments were done in p53-null H1299 cells and p53 expression (WTp53, DNp53:WTp53, or WTp53:WTp53) was induced by transient transfection. After normalization for total p53 protein levels, gene expression changes were assessed using microarrays, which measure steady-state mRNA levels, and comparative metabolomics experiments were completed. We now seek to employ more rigorous and informative next generation methods to address how the naturally occurring DNp53 isoform alters WTp53 function. Instead of transfection into p53-null cells, we will use genome-edited CRISPR- Cas9 knock-in cell lines (MCF10a, which normally express WTp53) in which DNp53:WTp53, WTp53:WTp53, or WTp53 (control) have been inserted into the native TP53 locus. Instead of microarrays, steady-state mRNA levels will be assessed with RNA-Seq (Aim 2), and we will assess global transcriptional changes using PRO- Seq (Aim 1)?which directly and precisely measures nascent transcription?and assess potential changes in p53 genomic occupancy using ChIP-Seq (Aim 1). One shared method (i.e. used in the 2013 study and also proposed here) will be the metabolomics analyses (Aim 2), which will be completed by Metabolon Inc., as before. In addition to application of cutting-edge methods and our innovative strategy to define the biological effects of DNp53 specifically, a key aspect of this study is chronic, yet transient induction of p53 over time (Aim 2). Brief periods of cell stress (i.e. p53 induction) have been shown to elevate human ?Np53 protein levels, and such transient increases are a basic feature in our experimental design. If successful, these studies will identify key transcriptional and/or metabolic pathways that are altered by DNp53:WTp53 expression. Given its established links to mammalian aging, this targeted project may reveal basic mechanisms by which DNp53 (a naturally occurring isoform) might help regulate normal, physiological aging.